HIV-1 entry into target cells is mediated by the successive interaction of the envelope glycoprotein gp120 with CD4 and a co-receptor belonging to the seven trans-membrane G protein-coupled chemokine receptor family (Berger et al. Ann. Rev. Immunol. 17:657, 1999). Binding of gp120 to CD4 exposes or creates a co-receptor binding site on gp120 (Trkola et al. Nature 384:184, 1996, Wu et al. Nature, 384:179, 1996). CCR5 and CXCR4 are the most physiologically relevant and widely used HIV-1 co-receptors (Zhang and Moore, J. Virol. 73:3443, 1999). CCR5 mediates the entry of R5 isolates and CXCR4 mediates the entry of X4 isolates. R5X4 isolates are able to exploit both co-receptors (Berger et al., Ann. Rev. Immunol. 17:657, 1999). It has been demonstrated that specific amino acids including acidic residues and tyrosines located within the CCR5 amino-terminal domain (Nt, amino acids 2-31) are essential for CCR5-mediated fusion and entry of R5 and R5X4 HIV-1 strains (Dragic et al. J. Virol. 72:279, 1998; Rabut et al. J. Virol. 72:3464, 1998; Farzan et al. J. Virol. 72:1160, 1998; Dorantz et al. J. Virol. 71:6305, 1997). More recently, Farzan et al. demonstrated that tyrosine residues in the CCR5 Nt are sulfated (Farzan et al. Cell 96:667, 1999)
Inhibition of cellular sulfation pathways, including tyrosine sulfation, by sodium chlorate decreased the binding of a gp120/CD4 complex to CCR5+ cells (Farzan et al. Cell 96:667, 1999). A number of prior reports had implicated a role for sulfate moieties in HIV-1 entry. Several sulfated compounds, such as dextran sulfate, can inhibit HIV-1 entry by associating with CD4 or gp120 (Baeuerle and Huttner J. Cell Biol 105:2655, 1987; Baba et al. Proc. Natl. Acad. Sci. USA 85:6132, 1998). Sulfated proteoglycans have been shown to bind to HIV-1 gp120 at or near its third variable (V3) loop, which also determines co-receptor usage (Roderiquez et al. J. Virol. 69:2233, 1995; Hwang et al. Science 253:71, 1991). It is therefore conceivable that sulfo-tyrosines in the CCR5 Nt also interact with gp120, increasing its affinity for CCR5. The reduction in gp120/CD4 binding caused by the pre-treatment of target cells with sodium chlorate, however, cannot be formally attributed to a reduction in CCR5 tyrosine sulfation since chlorate can inhibit the sulfation of both tyrosines and proteoglycans.
The region of the CCR5 Nt spanning amino acids 2-18 contains residues that are critically important for viral entry (Dragic et al. J. Virol. 72:279, 1998; Rabut et al. J. Virol. 72:3464, 1998; Farzan et al. J. Virol. 72:1160, 1998; Dorantz et al. J. Virol. 71:6305, 1997). We previously demonstrated that tyrosines at positions 3, 10 and 14 were required for optimal co-receptor function, whereas the Tyr15Phe substitution had little effect on entry (Rabut et al. J. Virol. 72:3464, 1998). Taken together, these findings suggested that HIV-1 entry may be critically dependent upon sulfation of Tyr-3, -10 and -14, but not Tyr-15. We therefore explored the role of sulfo-tyrosines in positions 3, 10 and 14 by synthesizing peptides corresponding to amino acids 2-18 of the CCR5 Nt and carrying different tyrosine modifications. We first tested the ability of the Nt peptides to inhibit binding of gp120/CD4 complexes and anti-CCR5 MAbs to CCR5+ cells. The specific association of certain peptides with gp120/sCD4 complexes or with anti-CCR5 MAbs was further confirmed by surface plasmon resonance (BIAcore) analysis. Inhibition of HIV-1 entry by the CCR5 Nt peptides was also tested. Our results suggest that amino acids 2-18 of the CCR5 Nt compose a gp120-binding site that determines the specificity of the interaction between CCR5 and gp120s from R5 and R5X4 isolates. Post-translational sulfation of the tyrosine residues in the CCR5 Nt is required for gp120 binding and may critically modulate the susceptibility of target cells to HIV-1 infection in vivo.
CCR5's normal physiologic activities involve binding and transducing signals mediated by CC-chemokines, including RANTES, MIP-1α and MIP-1β, which direct activation and trafficking of T cells and other inflammatory cells. As such, CCR5 plays an important role in mediating the inflammatory reaction of diseases such as rheumatoid arthritis and multiple sclerosis. The synovial fluid of rheumatoid arthritis patients is highly enriched in CCR5-expressing T cells (Qin et al. J Clin Invest 101:746, 1998), and CCR5 is the predominant CC chemokine receptor expressed on T cells in the rheumatoid synovium (Gomez-Reino et al. Arthritis Rheum 42:989, 1999). Similarly, infiltration by CCR5-expressing cells is characteristic of plaque lesions in patients with multiple sclerosis (Balashov et al. Proc Natl Acad Sci USA 96:6873, 1999) Such observations provide a rationale for the use of agents that block CCR5 for therapy of inflammatory/autoimmune diseases, including but not limited to arthritis, multiple sclerosis, asthma, psoriasis, autoimmune diabetes, transplant rejection, and atherosclerosis.